Cell death is an essential event in the development and functioning of multicellular organisms, in adult organisms, cell death plays a complementary role to mitosis in the regulation of cell populations. The pathogenesis of numerous diseases involves failure of tissue homeostasis which is presumed to be linked with cytotoxic injury or loss of normal control of cell death.
There exist two major types of cell death, necrosis and apoptosis, marked by different morphological and molecular characteristics (Kerr et al., Br. J. Cancer. 26: 239-257, 1972; Umansky, J. Theor. Biol. 97: 591-602, 1982; Umansky and Tomei, Adv Pharmacol. 41: 383-407, 1997; Ameisen, Cell Death Differ. 11: 4-10, 2004; Lockshin and Zakeri, Int J Biochem Cell Biol. 36: 2405-19, 2004; and Kroemer, et al., Cell Death and Differentiation 12: 1463-1467, 2005). Necrosis is considered to be catastrophic metabolic failure resulting directly from severe molecular and/or structural damage and leads to inflammation and secondary damage to surrounding cells. Apoptosis, also termed programmed cell death, is a much more prevalent biological phenomenon than necrosis and can be induced by specific signals such as hormones, cytokines, by absence of specific signal such as growth or adhesion factors, or by molecular damage that does not cause catastrophic loss of integrity. Apoptosis is a result of an active cellular response involving initiation of an orderly and specific cascade of molecular events. Apoptosis leads to the appearance of distinctive chromatin condensation and margination, nuclear fragmentation, cell shrinkage, and membrane blebbing. Enzymatic internucleosomal fragmentation of nuclear DNA is a hallmark of apoptosis, although some cells die by apoptosis without internucleosomal DNA cleavage (Umansky et al., Biochim Biophys Acta. 655: 9-17, 1981; rends et al., Am J. Pathol. 136: 593-608, 1990; and Zimmermann et al., Pharmacol Ther. 92: 57-70, 2001). Other, more rare forms of cell death, characterized by specific morphology, for example the so-called autophagic cell death, have also been described.
It is well known that apoptosis, or programmed cell death, which is a major form of cell death in multicellular organisms, is accompanied by internucleosomal fragmentation of nuclear DNA. This DNA originates from all cells undergoing apoptosis and thus from all tissues throughout the body. Many laboratories have demonstrated that in humans a portion of this DNA appears in blood (Lo et al., Ann NY Acad Sci. 945: 1-7, 2001; Lichtenstein et al., Ann NY Acad Sci. 945: 239-249, 2001; Taback and Hoon, Curr. Opin Mol Ther. 6: 273-278, 2004; and Bischoff et al., Hum Reprod Update. 8: 493-500, 2002). It has also been shown that this fragmented DNA crosses the kidney barrier (Transrenal DNA or Tr-DNA) and can be detected in the urine (Botezatu et al., Clin Chem. 46:1078-1084, 2000; Su et al., J Mol Diagn. 6:101-107, 2004; and Su et al., Ann NY Acad Sci. 1022: 81-89, 2004).
Both cell-free plasma DNA and Transrenal-DNA (Tr-DNA) have been used as diagnostic tools when the diagnostic marker is the presence of specific, known sequences different from bulk genomic DNA. For example, detection of tumor-specific DNA that results from characteristic mutations can be used for tumor diagnostics, detection of male Y chromosome-specific sequences in urine or blood of a pregnant woman can be used to determine the male gender of the fetus and detection of mutations characteristic of inherited disease can provide a tool for prenatal genetic testing (Chan and Lo, Semin Cancer Biol. 12: 489-496, 2002; Goessl, Expert Rev Mol Diagn. 3: 431-442, 2003; Su et al., J Mol Diagn. 6: 101-107, 2004; Wataganara and Bianchi, Ann NY Acad Sci. 1022: 90-99, 2004; Botezatu et al., Clin Chem. 46: 1078-1084, 2000; and Ding et al., Proc Natl Acad Sci USA. 101: 10762-10767, 2004).
The fate of RNA from dying cells, in particular the mechanisms of its degradation, is much less investigated. However, it is known that fetal RNA can be detected in plasma of pregnant women and RNA with tumor-specific mutations is detectable in plasma of patients with different types of cancer (Tsui et al., Ann NY Acad Sci. 2006; 1075:96-102; Lo and Chiu, Nat Rev Genet. 8: 71-77, 2007; and Tsang and Lo, Pathology 39: 197-207, 2007).
These specific nucleic acid biomarkers are often very short and their concentration in body fluids is usually low, especially if a test addresses an early stage of pregnancy or a disease. Thus, new methods are needed to detect these sensitive biomarkers. The present invention addresses this need in the art.